An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers

Abstract

Previously, we showed that caveolin-1 (Cav-1) expression is down-regulated in human breast cancer-associated fibroblasts. However, it remains unknown whether loss of Cav-1 occurs in the breast tumor stroma in vivo. Here, we immunostained a well-annotated breast cancer tissue microarray with antibodies against Cav-1 and scored its stromal expression. An absence of stromal Cav-1 was associated with early disease recurrence, advanced tumor stage, and lymph node metastasis, resulting in a 3.6-fold reduction in progression-free survival. When tamoxifen-treated patients were selected, an absence of stromal Cav-1 was a strong predictor of poor clinical outcome, suggestive of tamoxifen resistance. Interestingly, in lymph node-positive patients, an absence of stromal Cav-1 predicted an 11.5-fold reduction in 5-year progression-free survival. Clinical outcomes among patients positive for HER2, and patients triple-negative for estrogen receptor, progesterone receptor and HER2, were also strictly dependent on stromal Cav-1 levels. When our results were adjusted for tumor and nodal staging, an absence of stromal Cav-1 remained an independent predictor of poor outcome. Thus, stromal Cav-1 expression can be used to stratify human breast cancer patients into low-risk and high-risk groups, and to predict their risk of early disease recurrence at diagnosis. Based on related mechanistic studies, we suggest that breast cancer patients lacking stromal Cav-1 might benefit from anti-angiogenic therapy in addition to standard regimens. We conclude that Cav-1 functions as a tumor suppressor in the stromal microenvironment.

Figure 1

Stromal caveolin-1 expression in human breast cancers and normal tissue. Breast tumor microarray samples were immunostained with antibodies directed against Cav-1 and subjected to scoring, as detailed in Materials and Methods. Representative examples are shown. Panels A and B show Cav-1 expression in the stroma of invasive ductal carcinomas. Panel C shows an absence of Cav-1 in the neoplastic stroma; however, endothelial cells still remain Cav-1 positive (see arrowheads). Panel D depicts normal human breast tissue (TDLUs; terminal ductal lobular units) for comparison purposes. Note that the mammary intralobular stroma, the vasculature, and myo-epithelial cells are normally Cav-1 positive.

Figure 2

Kaplan-Meier curves of progression-free survival for patients with and without tamoxifen treatment. Left panel: Note that an absence of stromal Cav-1 immunostaining predicts poor clinical outcome in Tamoxifen-treated patients, suggestive of tamoxifen-resistance. Right panel: Virtually identical results were obtained with patients that did not receive tamoxifen. In both panels, 5-year PFS is indicated by an arrow. Tamoxifen-treated (P = 4.61 × 10⁻⁵, log-rank test); No tamoxifen (P = 7.74 × 10⁻⁵, log-rank test).

Figure 3

Epithelial Cav-1 expression is not a predictor of progression-Free survival. The status of stromal and epithelial Cav-1 was independently scored in the same total patient population for direct comparison. Note that only stromal Cav-1 is a predictor of clinical outcome (P = 1.77 × 10⁻⁹, log-rank test), in a total population of 125 breast cancer patients. 5-year PFS is indicated by an arrow. The status of epithelial Cav-1 is also shown. n.s., denotes not significant.

Figure 4

Kaplan-Meier curves of progression-free survival in ER-positive patients. Note that an absence of stromal Cav-1 immunostaining also predicts poor clinical outcome in ER-positive patients (P = 5.94 × 10⁻⁷, log-rank test), which represents a total of 80 breast cancer patients. 5-year PFS is indicated by an arrow. The status of epithelial Cav-1 is shown for comparison. n.s., denotes not significant.

Figure 5

Kaplan-Meier curves of PFS in PR-positive patients. Note that an absence of stromal Cav-1 immunostaining also predicts poor clinical outcome in PR-positive patients (P = 1.18 × 10⁻⁵, log-rank test), which represents a total of 65 breast cancer patients. 5-year PFS is indicated by an arrow. The status of epithelial Cav-1 is shown for comparison. n.s., denotes not significant.

Figure 6

Kaplan-Meier curves of PFS in HER2-positive patients. Note that an absence of stromal Cav-1 immunostaining also predicts poor clinical outcome in HER2-positive patients (P = 7.97 × 10⁻³, log-rank test), which represents a total of 32 breast cancer patients. 5-year PFS is indicated by an arrow. The status of epithelial Cav-1 is shown for comparison. n.s., denotes not significant.

Figure 7

Kaplan-Meier curves of PFS in triple-negative patients. Note that an absence of stromal Cav-1 immunostaining also predicts poor clinical outcome in triple-negative (ER−/PR−/HER2−) patients (P = 2.01 × 10⁻², log-rank test), even though this subset of the patient population is small (16 patients). 5-year PFS is indicated by an arrow. The status of epithelial Cav-1 is shown for comparison. n.s., denotes not significant.

Figure 8

Kaplan-Meier curves of PFS in LN-negative and positive patients. Note that in both LN(−) and LN(+) patients, an absence of stromal Cav-1 still remains a significant predictor of progression-free outcome. However, the results were most dramatic in LN(+) patients, where an absence of stromal Cav-1 is associated with an ∼11.5-fold reduction in 5-year progression-free survival. There were 50 patients in the LN(−) group and 54 patients in the LN(+) group. P values are as shown. 5-year PFS is indicated by an arrow.